PCR Genotyping from Tail Biopsy Genomic DNA
This is a dirty prep, but less labor intensive for standard genotyping. The tail biopsy should be taken when the mouse is ~14-15 days old, the genomic DNA extracted is of higher quality than older mice that have more cartilage and bone. The yield is 50-100 ug.
1. Freshly prepare PCR/Proteinase K digestion solution
1. 1 mg/ml proteinase K (from frozen stock of 10 mg/ml, store small aliquots to minimize freeze/thaws)
2. 1X PCR Buffer
3. 1% Triton X-100 (from10% stock)
2. Aliquot 100ul of this solution into numbered eppendorf tubes
3. Use clean sharp scissors cut 2-5 mm piece of tail and place in digestion solution. Clean scissors with a 10% bleach solution followed by a70% ethanol solution to prevent contamination between samples
4. Digest tissue at 55 C for 2-3 hours, or until completely dissociated. Gently rocking helps speed digestion
5. Inactivate the proteinase K by heating samples to 95 C for 10 min.
6. At this point the DNA sample can be stored frozen or used directly for a PCR assay. Use 5-10 ul of sample for your template, ~1/5 of your volume for PCR.
PCR Buffer:
67 mM Tris HCl, pH 8.8
16.6 Ammonium sulfate
6.7 mM Magnesium chloride
5 mM B-Mercaptoethanol